Kidney-targeted gene transfer has the potential to be one of the most important tools for broadening the understanding of renal disease processes of genetic renal diseases, glomerulonephritis, diabetic nephropathy, acute and chronic renal failure, renal graft and the like, or for revolutionizing treatments of these renal diseases.
Although various methods for kidney-targeted transfer of a gene and an expression thereof have been so far reported, the amount of expressed protein and duration of the expression have been insufficient for a therapeutic use (Gene Ther. 4, 426-431, 1997; J. Clin. Invest. 101, 1320-1325, 1998; Kidney Int. 57, 1973-1980, 2000; Hum. Gene Ther. 8, 1243-1251, 1997; Gene Ther. 7, 279-285, 2000; Biochem. Biophys. Res. Commun. 186, 129-34, 1992; J. Clin. Invest. 92, 2597-2601, 1993; Biochem. Biophys. Res. Commun. 206, 525-532, 1995).
Further, as a vector for transferring an exogenous gene (therapeutic gene), viral vectors (adenovirus vector, herpesvirus vector and the like) are mainly used in view of the transfer efficiency. The use of these viral vectors are however still problematic in securing safety, excluding antigenicity and the like.
Moreover, regarding a sort of nonviral vector, methods of improving the transfer efficiency using synthetic polymers, such as a complex with a cationic liposome (lipoplex) and a complex with a cationic polymer (polyplex), have been also proposed. However, in case of using these synthetic polymers as a vector, it must be concerned with a problem such as disorder of an immune system resulted from the adjuvant function thereof.
Meanwhile, a method has been proposed, in which a recombinant plasmid vector is directly injected into the skeletal muscle, skin or cardiac muscle of mammals and a protein or a peptide is produced in vivo by expression of the gene that is encoding thereof and incorporated in the plasmid vector. This method is based on interesting characteristics of plasmid DNA (naked DNA) reported by Wolff et al. That is, Wolff et al. have reported that; when a plasmid vector recombined with genes encoding various enzymes (β-galactosidase, luciferase and the like) was directly injected into the muscle of mammals, the recombinant plasmid DNA kept its existence as an episome (extrachromosomal element) in the muscle cells over a long period of time (for several months) without being replicated or incorporated into a host genome, and it was observed in the meantime that those enzymes encoded by insert genes was continuously expressed (Science, 247:1465-1468, 1990). Since then, the gene transfer and expression method by intramuscular injection of this plasmid vector has been expected to be a main stream of future gene therapy that replaces the recombinant viral vector or the like, and its application range has been variously studied. For example, a method had been developed, in which an antigen is expressed in muscle cells by means of intramuscularly injection of a plasmid vector with an incorporated DNA fragment encoding the antigenic protein, and immunological resistance of host is acquired stimulation of the antigen-specific immune response therein. This method is named “DNA vaccine”, and excellent in cost and safety in comparison with ordinary inactivated or attenuated virus vaccines. Thus, it attracts much interest as a vaccine in the next generation. In addition to the use as a DNA vaccine for acquiring an immunological resistance, a method of directly transferring the recombinant plasmid vector was shown to be effective for controlling a systemic function with a physiologically active substance or the like. For example, it was reported that a recombinant plasmid vector encoding cytokines (IL-2, IL-4, TGFβ1) was intramuscularly injected into mice, whereby the cytokines expressed in the muscle cells was subjected to systemic circulation and function systemically (Pro. Natl. Acad. Sci. USA, 93:10876-10880, 1996). Incidentally, one of the inventors of this application invented a method using electroporation as an effective approach to transfer a recombinant plasmid vector into cells, and the invention has already been applied for patent (official gazette of JP-A-2000-004715).
As mentioned above, the gene transfer using the recombinant plasmid vector (naked DNA) has been so far employed as a method of directly transferring a vector into tissue cells such as muscle cells, and has never been taken advantage of as a vector to transfer an exogenous gene into in vivo organs.
This invention aims to provide a novel method that enables long-term expression of a transgene using a recombinant plasmid vector as a kidney-targeted gene transfer vector.
Further, it was known that peritubular capillaries (PTC) of the kidney, which comprise a network of interstitial vessels that connects veins at each cortical level, play a major role in maintaining renal function and hemodynamics [The kidney Vol. 1, 6th edn. (ed. Brenner, B. M.) 277-318, W. B. Saunders, Philadelphia, USA, 2000]. Progressive tubulointerstitial fibrosis, which is accompanied by the loss of PTC or tubules typifies, was known as common symptom in all progressive renal diseases (J. Am. Soc. Nephrol. 7, 2495-2508, 1996). The severity of chronic tubulointerstitial changes, rather than that of glomerular damage, was strongly related with decline in renal function and long-term prognosis (Lancet. 2, 363-366, 1968; Hum. Pathol. 1, 631-641, 1970; Nephrol. Dial. Transplant, 5, 889-893, 1990; Kidney Blood Press. Res. 19, 191-195, 1996; J. Am. Soc. Nephrol. 9, 231-242, 1998; J. Am. Soc. Nephrol. 11, 47-56, 2000). Moreover, it was known that an endothelium of the PCT is one of the main targets of acute and chronic renal transplant rejection (17. J. Am. Soc. Nephrol. 10, 2208-2214, 1999; Lab. Invest. 80, 815-830, 2000; J. Am. Soc. Nephrol. 12, 574-582, 2001).
Accordingly, the invention of this application more specifically aims to express an exogenous gene in fibroblasts adjacent to the endothelium of PTC over a long period of time.